Methods for biosynthesizing 1,3 butadiene

ABSTRACT

This document describes biochemical pathways for producing butadiene by forming two vinyl groups in a butadiene synthesis substrate. These pathways described herein rely on enzymes such as, inter alia, a decarboxylating thioesterase, cytochrome P450, or dehydratases for the final enzymatic step.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/334,190 filedJul. 17, 2014, which claims priority from U.S. Provisional ApplicationSer. No. 61/856,154, filed Jul. 19, 2013. The contents of the priorapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing 1,3-butadiene, andmore particularly to synthesizing 1,3-butadiene using one or moreisolated enzymes such as dehydrogenases, dehydratases, decarboxylatingthioesterases, decarboxylating cytochrome P450s or using recombinanthost cells expressing one or more of such enzymes.

BACKGROUND

1,3-Butadiene (sometimes referred to herein as “butadiene”) is animportant monomer for the production of synthetic rubbers includingstyrene-butadiene-rubber (SBR), polybutadiene (PB), styrene-butadienelatex (SBL), acrylonitrile-butadiene-styrene resins (ABS), nitrilerubber, and adiponitrile. Adiponitrile is used in the manufacture ofNylon-66 (White, Chemico-Biological Interactions, 2007, 166, 10-14).

Butadiene is typically produced as a co-product from the steam crackingprocess, distilled to a crude butadiene stream, and purified viaextractive distillation (White, Chemico-Biological Interactions, 2007,166, 10-14).

On-purpose butadiene has been prepared among other methods bydehydrogenation of n-butane and n-butene (Houdry process); and oxidativedehydrogenation of n-butene (Oxo-D or O-X-D process) (White,Chemico-Biological Interactions, 2007, 166, 10-14).

Industrially, 95% of global butadiene production is undertaken via thesteam cracking process using petrochemical-based feedstocks such asnaphtha. Production of on-purpose butadiene is not significant, giventhe high cost of production and low process yield (White,Chemico-Biological Interactions, 2007, 166, 10-14).

Given reliance on petrochemical feedstocks and, for on-purposebutadiene, energy intensive catalytic steps; biotechnology offers analternative approach via biocatalysis. Biocatalysis is the use ofbiological catalysts, such as enzymes, to perform biochemicaltransformations of organic compounds.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing intermediates, in particularbutadiene, wherein the methods are biocatalyst based (Jang et al.,Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

The generation of two vinyl groups into medium carbon chain lengthenzyme substrates is a key consideration in synthesizing butadiene viabiocatalysis processes.

There are no known enzyme pathways leading to the synthesis of butadienein prokaryotes or eukaryotes. Three potential pathways have beensuggested for producing 1,3-butadiene from biomass-sugar: (1) fromacetyl-CoA via crotonyl-CoA; (2) from erythrose-4-phosphate; and (3) viaa condensation reaction with malonyl-CoA and acetyl-CoA. However, noinformation using these strategies has been reported (Jang et al.,Biotechnology & Bioengineering, 2012, 109(10), 2437-2459).

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing medium length(e.g., C5) chain carbon metabolites, in which two vinyl groups can beformed, leading to the synthesis of butadiene. These pathways, which aredescribed herein, rely on enzymes such as sulphotransferases anddecarboxylating thioesterases, decarboxylating cytochrome P450s anddehydratases for the final enzymatic step.

It was not previously known that enzymes capable of forming two terminalvinyl groups in a medium chain carbon metabolite existed or could beproduced for the synthesis of butadiene.

Thus, in one aspect, this document provides enzymes that can convertbutadiene synthesis substrates into butadiene. As used herein, the term“butadiene synthesis substrate” refers to a substrate for which anenzyme can catalyze a reaction that results directly in 1,3-butadiene orin a product that, after one or more enzyme-catalyzed reactions, isconverted to 1,3-butadiene. Relevant enzymes are those disclosed hereinas having this activity.

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in pent-2-enoyl-[acp] to produce2,4-pentadienoyl-[acp]. (FIG. 2).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in lactoyl-CoA,3-hydroxypropionyl-CoA, or propanoyl-CoA to produce propenoyl-CoA. (FIG.3).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in 5-hydroxypentanoyl-CoA (via5-hydroxy-pent-2-enoyl-CoA as intermediate) to produce2,4-pentadienoyl-CoA. (FIG. 4).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in 4-hydroxy-pent-2-enoyl-CoA toproduce pent-2,4-dienoyl CoA (2,4-pentadienoyl-CoA). (FIG. 6).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in 2-butanol (butan-2-ol),2-buten-1-ol or 2-buten-1-ol diphosphate to produce 3-buten-2-ol. (FIG.5).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is formed in (R) 3-hydroxypent-4-enoyl-[acp] or (R)3-hydroxypent-4-enoyl-CoA by sulphotransferase followed bydecarboxylating thioesterase activity ((i) and (vi) in FIG. 7).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is formed in (R) 3-hydroxypent-4-enoyl-[acp] or (R)3-hydroxypent-4-enoyl-CoA by phosphotransferase followed by thioesteraseactivity ((ii) and (iii) in FIG. 7).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is formed in pent-4-enoyl-CoA by thioesterase activityfollowed by activity of a decarboxylating cytochrome P450 in the CYP152family ((v) in FIG. 7).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is formed in 3-buten-2-ol by linalool dehydratase classifiedin EC 4.2.1.127 or a dehydratase classified under EC 4.2.1.—(such as oneisolated from species such as Aquincola tertiaricarbonis or Methylibiumpetroleiphilum PM1) ((vi) in FIG. 7).

In one aspect, this document features a method for the biosynthesis ofbutadiene. The method includes forming two terminal vinyl groups in abutadiene synthesis substrate. A first vinyl group can be enzymaticallyformed in the butadiene synthesis substrate to produce a compoundselected from the group consisting of 2,4-pentadienoyl-[acp],propenyl-CoA, 2,4-pentadienoyl-CoA, 3-buten-2-ol and pent-4-enoyl-CoA

In one aspect, 2,4-pentadienoyl-[acp] can be produced by forming a firstvinyl group in pent-2-enoyl-[acp] using an acyl-[acp]dehydrogenase suchas encoded by tcsD (FIG. 2). The pent-2-enoyl-[acp] can be produced byconverting (R)-3-hydroxypentanoyl-[acp] using a3-hydroxyacyl-[acp]dehydratase classified under EC 4.2.1.59 such asencoded by fabZ (FIG. 2). The (R)-3-hydroxypentanoyl-[acp] can beproduced by converting (R)-3-oxopentanoyl-[acp] using a3-oxoacyl-[acp]reductase classified in EC 1.1.1.100 such as encoded byfabG or AnlG (FIG. 2). The (R)-3-oxopentanoyl-[acp] can be produced byconverting propanoyl-CoA using a β-ketoacyl-[acp]synthase such asencoded by tcsA and tcsB (FIG. 2). The propanoyl-CoA can be produced viaa number of pathways (FIG. 1).

In one aspect, propenoyl-CoA can be produced by forming a first vinylgroup in (i) 3-hydroxypropionyl-CoA using a 3-hydroxypropionyl-CoAdehydratase classified in EC 4.2.1.116, (ii) propanoyl-CoA using aacyl-CoA dehydrogenase classified in EC 1.3.8.-(1,7) or a2-methylacyl-CoA dehydrogenase classified in EC 1.3.99.12 or (iii)lactoyl-CoA using a lactoyl-CoA dehydratase classified in EC 4.2.1.54(FIG. 3).

The propanoyl-CoA can be produced via a number of pathways (FIG. 1).

The 3-hydroxypropionyl-CoA can be produced by converting3-hydroxypropionate using 3-hydroxyisobutyryl-CoA hydrolase classifiedin EC 6.2.1.—(FIG. 3). The 3-hydroxypropionate can be produced byconverting malonate semialdehyde using 3-hydroxyproprionatedehydrogenase classified in EC 1.1.1.59 (FIG. 3). The malonatesemialdehyde can be produced by converting malonyl-CoA using malonyl-CoAreductase classified in EC 1.2.1.75 (FIG. 3).

The lactoyl-CoA can be produced by converting L-lactate usingpropionate-CoA transferase classified in EC 2.8.3.1 (FIG. 3). TheL-lactate can be produced by converting pyruvate using L-lactatedehydrogenase classified in EC 1.1.1.27 (FIG. 3).

In one aspect, the 2,4-pentadienoyl-CoA can be produced by forming afirst vinyl group in 5-hydroxypentanoyl-CoA using a5-hydroxypentanoyl-CoA dehydratase classified in EC 4.2.1.—(FIG. 4)(such as one isolated from Clostridium viride). The5-hydroxypentanoyl-CoA can be produced by converting 5-hydroxypentanoateusing 5-hydroxypentanoyl-CoA CoA-transferase classified in EC 2.8.3.14(FIG. 4). The 5-hydroxypentanoate can be produced by converting5-oxopentanoic acid using a 5-hydroxypentanoate dehydrogenase (FIG. 4)(such as one encoded by cpnD). The 5-oxopentanoic acid can be producedby converting 5-aminovalerate (5-aminovaleric acid) using5-aminovalerate transaminase classified in EC 2.6.1.48 (FIG. 4). The5-aminovalerate can be produced via a number of pathways (FIG. 4).

In one aspect, 2,4-pentadienoyl-CoA (pent-2,4-dienoyl-CoA) can beproduced by forming a first vinyl group in 4-hydroxypent-2-enoyl-CoAusing a dehydratase such as linalool dehydratase classified in EC4.2.1.127 or a dehydratase classified under EC 4.2.1.—(such as oneisolated from species such as Aquincola tertiaricarbonis or Methylibiumpetroleiphilum PM1) (FIG. 6). The 4-hydroxypent-2-enoyl-CoA can beproduced by converting 4-hydroxypentanoyl-CoA using a reversibletrans-2-enoyl-CoA reductase such as one classified under EC1.3.1.—(e.g., EC 1.31.8 or EC 1.3.1.38) or EC 1.3.1.44 (e.g., oneencoded by ter or tdter) (FIG. 6). The 4-hydroxypentanoyl-CoA can beproduced by converting levulinyl-CoA using a secondary alcoholdehydrogenase classified under EC 1.1.1.B4 or a 3-hydroxybutanoateoxidoreductase homologue classified under EC 1.1.1.30. (FIG. 6). Thelevulinyl-CoA can be produced by converting levulinic acid using aCoA-ligase classified in EC 6.2.1.-. (FIG. 6).

In one aspect, the pent-4-enoyl-CoA can be produced by forming a firstvinyl group into 4-hydroxypentanoyl-CoA using a dehydratase such aslinalool dehydratase classified in EC 4.2.1.127 or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1) (FIG. 6).The 4-hydroxypentanoyl-CoA can be produced by converting levulinyl-CoAusing a secondary alcohol dehydrogenase classified under EC 1.1.1.B4 ora 3-hydroxybutanoate oxidoreductase homologue classified under EC1.1.1.30. (FIG. 6). The levulinyl-CoA can be produced by convertinglevulinic acid using a CoA-ligase classified under EC 6.2.1.-. (FIG. 6).

In one aspect, the 3-buten-2-ol can be produced by forming a first vinylgroup in (i) butan-2-ol using a desaturase or a monooxygenase such asencoded by MdpJ, (ii) 2-buten-1-ol using an isomerase (classified inEC5.4.4.—), (iii) 2-buten-1-ol diphosphate using a 2-methyl-3-buten-2-olsynthase (such as that encoded by Tps-MBO1) or (iv) 3-buten-2-one usinga (R)-specific secondary alcohol dehydrogenase (classified inEC1.1.1.B4. (FIG. 5).

The butan-2-ol can be produced by converting butanone (butan-2-one)using a (R)-specific secondary alcohol dehydrogenase classified under EC1.1.1.B4 (FIG. 5). The butan-2-one can be produced by converting2,3-butanediol using a propanediol dehydratase classified under EC4.2.1.28 or converting 3-oxopentanoate using an acetoacetatedecarboxylase classified under EC 4.1.1.4 (FIG. 5). The 2,3 butanediolcan be produced by converting (R)-acetoin using a (R,R)-butanedioldehydrogenase classified under EC 1.1.1.4. (R)-acetoin can be producedby converting 2-acetolactate using an acetolactate decarboxylaseclassified under EC 4.1.1.5 (FIG. 5) The 2-acetolactate can be producedby converting pyruvate using an acetolactate synthase classified underEC 2.2.1.6 (FIG. 5). The 3-oxopentanoate can be produced by converting3-oxopentanoyl-CoA using a thioesterase classified under EC 3.1.2.-,such as the gene product of YciA, tesB, tesA or fadM (FIG. 5). The3-oxopentanoyl-CoA can be produced by converting propanoyl-CoA using aβ-ketothiolase classified under EC 2.3.1.16. The propanoyl-CoA can beproduced via a number of pathways (FIG. 1).

The 2-buten-1-ol can be produced by converting 2-buten-1-al using anallyl-alcohol dehydrogenase classified under EC 1.1.1.54 (FIG. 5). The2-buten-1-al can be produced by converting crotonic acid using along-chain-aldehyde dehydrogenase classified under EC 1.2.1.48 (FIG. 5).Crotonic acid can be produced by converting crotonyl-CoA using asuccinate-CoA ligase classified under EC 6.2.1.5 (FIG. 5).

The 2-buten-1-ol diphosphate can be produced by converting 2-buten-1-olphosphate using a phosphomevalonate kinase (classified under EC 2.7.4.2)or by converting 2-buten-1-ol using a diphosphotransferases such as athiaminediphosphokinase classified under (EC 2.7.6.2) (FIG. 5). The2-buten-1-ol phosphate can be produced by converting 2-buten-1-ol usingmevalonate kinase (classified under EC 2.7.1.36) (FIG. 5).

The 3-buten-2-one can be produced by converting 3-oxopent-4-enoate usingan acetolactate decarboxylase classified under EC 4.1.1.4 (FIG. 5). The3-oxopent-4-enoate can be produced by converting 3-oxopent-4-enoyl-CoAusing a thioesterase classified under EC 3.1.2.—such as the gene productof YciA, tesB, tesA or fadM (FIG. 5). The 3-oxopent-4-enoyl-CoA can beproduced by converting propenyl-CoA using a β-ketothiolase classifiedunder EC 2.3.1. such as EC 2.3.1.16. (FIG. 5).

The second vinyl group can be enzymatically formed in3-sulphoryl-pent-4-enoyl-[acp] by a decarboxylating thioesterase such asthat encoded by CurM TE ((i) in FIG. 7)

The second vinyl group can be enzymatically formed in3-phospho-pent-4-enoyl-[acp] by a decarboxylating thioesterase such asthat encoded by CurM TE ((ii) in FIG. 7).

The second vinyl group can be enzymatically formed in3-sulphoryl-pent-4-enoyl-CoA by a decarboxylating thioesterase ((iv) inFIG. 7).

The second vinyl group can be enzymatically formed in3-phospho-pent-4-enoyl-CoA by a decarboxylating thioesterase ((iii) inFIG. 7)

The second vinyl group can be enzymatically formed in pent-4-enoate bydecarboxylating cytochrome P450 in the CYP152 family ((v) in FIG. 7).

The second vinyl group can be enzymatically formed in 3-buten-2-ol bylinalool dehydratase (classified under EC 4.2.1.127) or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1) ((vi) inFIG. 7).

In any of the methods described herein, the method can be performedusing isolated enzymes, using cell lysates comprising the enzymes, orusing a recombinant host.

The recombinant host can be anaerobically, micro-aerobically oraerobically cultivated.

Recombinant host cells can be retained in ceramic hollow fiber membranesto maintain a high cell density during fermentation.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. For example, the biologicalfeedstock is, or derives from, monosaccharides, disaccharides,lignocellulose, hemicellulose, cellulose, levulinic acid, furfural,lignin, triglycerides such as glycerol and fatty acids, agriculturalwaste or municipal waste. The non-biological feedstock is, or derivesfrom, either natural gas, syngas, CO₂/H₂, methanol, ethanol,non-volatile residue (NVR) or caustic wash waste stream from cyclohexaneoxidation processes.

The host microorganism can be a prokaryote from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis.

The host microorganism can be a eukaryote from the genus Aspergillussuch as Aspergillus niger; from the genus Saccharomyces such asSaccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris;from the genus Yarrowia such as Yarrowia lipolytica; from the genusIssatchenkia such as Issathenkia orientalis; from the genus Debaryomycessuch as Debaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis.

In any of the recombinant hosts described herein, the enzymes catalyzingthe hydrolysis of propionyl-CoA and acetyl-CoA can be attenuated; theenzymes consuming propanoyl-CoA via the methyl-citrate cycle can beattenuated; the enzymes consuming propanoyl-CoA to pyruvate can beattenuated; the enzymes consuming propanoyl-CoA to malonyl-CoA can beattenuated; a feedback-resistant threonine deaminase can be geneticallyengineered into the host organism; the β-ketothiolases catalyzing thecondensation of acetyl-CoA to acetoacetyl-CoA such as the gene productsof AtoB or phaA can be attenuated; the polymer synthase enzymes in ahost strain that naturally accumulates polyhydroxyalkanoates can beattenuated; a gene encoding a phosphotransacetylase, such as pta, can beattenuated; a gene encoding an acetate kinase degrading propanoate, suchas ack, can be attenuated; a gene encoding the degradation of pyruvateto lactate can be attenuated; a gene encoding the degradation ofphophoenolpyruvate to succinate such as frdBC can be attenuated; a geneencoding the degradation of acetyl-CoA to ethanol such as adhE can beattenuated; the enzymes catalyzing anaplerotic reactions supplementingthe citric acid cycle intermediates can be amplified; the3′-phosphoadenosine 5′-phosphosulfate synthase (EC 2.7.7.4, EC 2.7.7.5)and 3′-phosphoadenosine 5′-phosphosulfate synthase (EC 2.7.1.25) can beconstitutively expressed; a primary amine oxidase (EC 1.4.3.21) anddecarboxylating cytochrome P450 can be expressed co-located or tethered;a puridine nucleotide transhydrogenase gene such as UdhA can beoverexpressed; a glyceraldehyde-3P-dehydrogenase gene such as GapN canbe overexpressed in the host organisms; a malic enzyme gene such as maeAor maeB is overexpressed in the host organism; a glucose-6-phosphatedehydrogenase gene such as zwf can be overexpressed in the hostorganism; a fructose 1,6 diphosphatase gene such as fbp can beoverexpressed in the host organism; the efflux of butadiene across thecell membrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membraneand/or the efflux of butadiene across the cell membrane to theextracellular media can be enhanced or amplified by geneticallyengineering an increase to any associated transporter activity forbutadiene; oxygenases degrading butadiene to toxic intermediates such as1,2-epoxy-3-butene and 1,2:3,4-diepoxybutane can be attenuated in thehost organism.

More specifically, this document provides a first method of butadienesynthesis; the method includes introducing a first vinyl group into afirst vinyl group acceptor compound using a dehydratase, adehydrogenase, a isomerase, a synthase or a desaturase.

In a second method of butadiene synthesis, the method includesintroducing a second vinyl group into a second vinyl group acceptorcompound using a decarboxylating thioesterase, a decarboxylatingcytochrome P450 or a dehydratase. The second method can further involve,prior to the introducing the second vinyl group, carrying out the firstmethod, i.e., introducing a first vinyl group into a first vinyl groupacceptor compound to produce the second vinyl group acceptor compound,using a dehydratase, a dehydrogenase, a isomerase, a synthase or adesaturase.

As used herein, a “first vinyl group acceptor compound” is a compound inany 1,3-butadiene synthetic pathway disclosed herein into which a firstvinyl group corresponding to one of two vinyl groups in 1,3-butadiene isfirst introduced and is retained in any subsequent intermediatecompounds in the pathway terminating in the production of 1,3-butadiene.Examples of first vinyl group acceptor compounds includepent-2-enoyl-[acp], 3-hydroxypropionyl-CoA, propanoyl-CoA, lactoyl-CoA,5-hydroxypentanoyl-CoA, butan-2-ol, 2-buten-1-ol, 2-buten-1-oldiphosphate, 4-hydroxy-pentanoyl-CoA, and 4-hydroxy pent-2-enoyl-CoA.

As used herein, a “second vinyl group acceptor compound” is a compoundin any 1,3-butadiene synthetic pathway disclosed herein that contains afirst vinyl group corresponding to one of two vinyl groups in1,3-butadiene and into which a second vinyl group corresponding to thesecond of the two vinyl groups in 1,3-butadiene is first introduced andis retained in any subsequent compounds in the pathway terminating inthe production of 1,3-butadiene. It is noted that the introduction ofthe second vinyl group is most commonly the last step in the pathway,i.e., the step in which the reaction product is 1,3-butadiene. Examplesof second vinyl group acceptor compounds include3-sulphorylpent-4-enoyl-[acp], 3-phosphopent-4-enoyl-[acp],3-phosphopent-4-enoyl-CoA, 3-sulphorylpent-4-enoyl-CoA, pent-4-enoate,and 3-buten-2-ol.

In the second method, the decarboxylating thioesterase introducing thesecond vinyl group can be an engineered enzyme having greater than 70%homology to the decarboxylating thioesterase from Lyngbya majuscula(CurM TE), Pseudomonas entomophila, H. ochraceum, Synechococcus PCC7002, Cyanothece PCC 7424 or Cyanothece PCC 7822. In addition, thedecarboxylating thioesterase introducing the second vinyl group cancatalyse the hydrolysis of either 3-sulphorylpent-4-enoyl-[acp],3-phosphopent-4-enoyl-[acp], 3-sulphorylpent-4-enoyl-CoA or3-phosphopent-4-enoyl-CoA. Moreover, in the second method, following theintroduction of the second vinyl group, the resulting compound canundergo spontaneous decarboxylation to butadiene. Also in the secondmethod, the decarboxylating cytochrome P450 introducing the second vinylgroup can be an engineered enzyme having greater than 70% homology tothe decarboxylating cytochrome P450 from Jeotgalicoccus sp. ATCC 8456.Furthermore in the second method, the dehydratase introducing the secondvinyl group can be an engineered enzyme having greater than 70% homologyto linalool dehydratase (EC 4.2.1.127) from Castellaniella defragrans.In the second method, the decarboxylating thioesterase can convert3-sulphorylpent-4-enoyl-[acp] or 3-phosphopent-4-enoyl-[acp] assubstrate to butadiene. Also, in the second method, the decarboxylatingcytochrome P450 can convert pent-4-enoic acid to butadiene and thehydrogen peroxide co-substrate required for the conversion ofpent-4-enoic acid to butadiene can optionally be provided by theactivity of a primary amine oxidase. Moreover in the second method, thedehydratase can convert 3-buten-2-ol to butadiene.

In the first method, whether carried out alone or prior to introducing asecond vinyl group, the dehydratase (EC 4.2.1.—) enzyme introducing thefirst vinyl group can be an engineered enzyme having greater than 70%homology to the 5-aminovaleryl-CoA dehydratase from C. viride, linalooldehydratase (EC 4.2.1.127) or a dehydratase (EC 4.2.1.—) from speciessuch as Aquincola tertiaricarbonis or Methylibium petroleiphilum PM1.Moreover, the dehydrogenase (e.g., an acyl-ACP dehydrogenase)introducing the first vinyl group can be an engineered enzyme havinggreater than 70% homology to the gene product of tcsD. In addition, inthe first method, whether carried out alone or prior to introducing asecond vinyl group, the desaturase/monooxygenase introducing the firstvinyl group can be an engineered enzyme having greater than 70% homologyto the gene product of MdpJ or cytochrome P450 CYT4 family. Furthermore,in the first method, whether carried out alone or prior to introducing asecond vinyl group, the synthase introducing the first vinyl group canbe an engineered enzyme have greater than 70% homology to2-methyl-3-buten-2-ol synthase encoded by Tps-MBOJ. Moreover, in thefirst method, whether carried out alone or prior to introducing a secondvinyl group, the isomerase introducing the first vinyl group can be anengineered enzyme having greater than 70% homology to the isomerase fromPseudomonas putida catalyzing the conversion of 2-methyl-3-buten-2-ol to2-methyl-3-buten-1-ol. Also, in the first method, whether carried outalone or prior to introducing a second vinyl group, the isomerase canconvert 2-buten-1-ol to 3-buten-2-ol.

Any of the methods can involve a fermentation process using a host cellexpressing an enzyme that catalyzes the introduction of a first vinylgroup, an enzyme that catalyzes the introduction of a second vinylgroup, or one or two enzymes that catalyze the introduction of a firstand a second vinyl group. The host cell can be either of a prokaryote ora eukaryote. The prokaryote can be of the genus Escherichia such asEscherichia coli; of the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; of thegenus Corynebacteria such as Corynebacterium glutamicum; of the genusCupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; ofthe genus Pseudomonas such as Pseudomonas fluorescens or Pseudomonasputida; of the genus Bacillus such as Bacillus subtillis; or of thegenus Rhodococcus such as Rhodococcus equi. Moreover, the eukaryote canbe of the genus Aspergillus such as Aspergillus niger; of the genusSaccharomyces such as Saccharomyces cerevisiae; of the genus Pichia suchas Pichia pastoris; of the genus Yarrowia such as Yarrowia lipolytica;of the genus Issatchenkia such as Issathenkia orientalis; of the genusDebaryomyces such as Debaryomyces hansenii; of the genus Arxula such asArxula adenoinivorans; or of the genus Kluyveromyces such asKluyveromyces lactis. The fermentation process can include anaerobic,micro-aerobic or aerobic cell cultivation. In these methods, cellretention strategies using, for example, ceramic hollow fibre membranescan be employed to achieve and maintain a high cell density duringfermentation. In addition, the principal carbon source fed to thefermentation can be derived from biological or non-biologicalfeedstocks. Biological feedstock can be, or can be derived from,monosaccharides, disaccharides, hemicellulose such as levulinic acid andfurfural, cellulose, lignocellulose, lignin, triglycerides such asglycerol and fatty acids, agricultural waste, or municipal waste.Non-biological feedstock can be, or can be derived from, natural gas,syngas, CO₂/H₂, methanol, ethanol, non-volatile residue (NVR), causticwash from a cyclohexane oxidation processes, or other waste stream fromthe chemical or petrochemical industries.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. The word “comprising” inthe claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of biochemical pathways leading to the productionof propanoyl-CoA from central metabolites.

FIG. 2 is a schematic of biochemical pathways leading to the productionof (R)-3-hydroxypent-4-enoyl-[acp] using propanoyl-CoA as a centralprecursor.

FIG. 3 is a schematic of biochemical pathways leading to the productionof 3-hydroxypent-4-enoyl-CoA using propenoyl-CoA as a central precursor.

FIG. 4 is a schematic of biochemical pathways leading to the productionof pent-4-enoyl-CoA using 5-aminovalerate (5-aminovaleric acid) as acentral precursor.

FIG. 5 is a schematic of biochemical pathways leading to the productionof 3-buten-2-ol using pyruvate or crotonyl-CoA as a central precursor.

FIG. 6 is a schematic of biochemical pathways leading to the productionof pent-4-enoyl-CoA or pent-2,4-enoyl-CoA as central precursor.

FIG. 7 is a schematic of biochemical pathways leading to the productionof butadiene using a sulphotransferase followed by a decarboxylatingthioesterase ((i) and (iv)), phosphotransferase followed bydecarboxylating thioesterase ((ii) and (iii)), a decarboxylatingcytochrome P450 in the CYP152 family (v), linalool dehydratase or adehydratase classified under EC 4.2.1.—(such as one isolated fromspecies such as Aquincola tertiaricarbonis or Methylibium petroleiphilumPM1) (vi).

DETAILED DESCRIPTION

The closest analogous compound synthesized by prokaryotes or eukaryotesis 2-methyl-1,3-butadiene (isoprene), given the short five carbon chainlength and two vinyl groups. Isoprene may be synthesised via two routesleading to the precursor dimethylvinyl-PP, viz. the mevalonate or thenon-mevalonate pathway (Kuzuyama, Biosci. Biotechnol. Biochem., 2002,66(8), 1619-1627).

The mevalonate pathway incorporates a decarboxylase enzyme, mevalonatediphosphate decarboxylase (hereafter MDD), that generates the firstvinyl-group in the precursors leading to isoprene (Kuzuyama, Biosci.Biotechnol. Biochem., 2002, 66(8), 1619-1627).

Enzymes with similar activity to mevalonate diphosphate decarboxylase(EC 4.1.1.33) may thus be earmarked as a candidate enzyme in thesynthesis of butadiene from non-native substrates.

The chain termination enzymatic reactions in the polyketide synthesis ofCuracin A involve sulphotransferase, encoded by CurM ST, andthioesterase, encoded by CurM TE, activity; mechanistically similar tothe activity associated with MDD, thus earmarking the enzymes ascandidates for the synthesis of butadiene from non-native substrates(Gehret et al., The Journal of Biological Chemistry, 2011, 286(16),14445-14454).

However, the activity of the sulphotransferase and thioesterase domainsin Curacin A biosynthesis has only been demonstrated for long (C12, C14)chain length substrate analogues (McCarthy et al., ACS Chem. Biol.,2012, 7, 1994-2003), teaching against using such sulphotransferase andthioesterase activity in the synthesis of butadiene from medium (e.g.,C5) chain length precursors.

The enzyme encoded by OleT_(JE) from the CYP152 cytochrome P450 family,introduces terminal vinyl groups into long chain fatty acids viadecarboxylation (Rude et al., Appl. Environ. Microbiol., 2011, 77(5),1718-1727).

The CYP152 fatty acid decarboxylase, OleT_(JE), may thus be earmarked asa candidate enzyme in the synthesis of butadiene from non-nativesubstrates.

Similar to the sulphotransferase and thioesterase activity terminatingCuracin A biosynthesis, OleT_(JE) has specificity for long chain lengthfatty acids (C18-C20) with low activity for C15 chain length fatty acids(Rude et al., Appl. Environ. Microbiol., 2011, 77(5), 1718-1727),teaching against using OleT_(JE) in the synthesis of butadiene frommedium (e.g., C5) chain length precursors.

In addition to decarboxylase activity, microorganisms can generate vinylgroups in metabolites typically via dehydratase, ammonia lyase ordesaturase activity. However, these enzyme activities rarely catalysethe formation of terminal vinyl groups. Dehydratases and ammonia lyasestypically accept fatty acid analogues that have activated hydrogen atomsor aromatic compounds, where the aromatic ring serves as an electronwithdrawing group. Desaturases predominate in fatty acid synthesis,generating unsaturated bonds at fixed non-terminal positions along longchain fatty acids. Therefore, the associated enzymatic activity of theseenzymes teaches against their use for the generation of terminal vinylgroups in short or medium chain carbon metabolites leading to thesynthesis of butadiene.

In particular, this document provides enzymes, non-natural pathways,cultivation strategies, feedstocks, host microorganisms and attenuationsto the host's biochemical network, which generate two terminal vinylgroups in four and five carbon chain metabolites leading to thesynthesis of 1,3 butadiene (sometimes referred to as “butadiene” herein)from central precursors or central metabolites. As used herein, the term“central precursor” is used to denote any metabolite in any metabolicpathway shown herein leading to the synthesis of butadiene. The term“central metabolite” is used herein to denote a metabolite that isproduced in all microorganisms to support growth.

As such, host microorganisms described herein can include endogenouspathways that can be manipulated such that butadiene can be produced. Inan endogenous pathway, the host microorganism naturally expresses all ofthe enzymes catalyzing the reactions within the pathway. A hostmicroorganism containing an engineered pathway does not naturallyexpress all of the enzymes catalyzing the reactions within the pathwaybut has been engineered such that all of the enzymes within the pathwayare expressed in the host. Within an engineered pathway, the enzymes canbe from a single source, i.e., from one species, or can be from multiplesources, i.e., different species. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL.

An the enzymes described herein that can be used for butadieneproduction can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acidsequence of the corresponding wild-type enzyme. The percent identity(homology) between two amino acid sequences can be determined asfollows. First, the amino acid sequences are aligned using the BLAST 2Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from the U.S. government's National Center for BiotechnologyInformation web site (ncbi with the extension.nlm.nih.gov of the worldwide web). Instructions explaining how to use the Bl2seq program can befound in the readme file accompanying BLASTZ. Bl2seq performs acomparison between two amino acid sequences using the BLASTP algorithm.To compare two amino acid sequences, the options of Bl2seq are set asfollows: —i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seq1.txt); —j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); —p is setto blastp; —o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\Bl2seq—i c:\seq1.txt—jc:\seq2.txt—p blastp—o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine, hemagluttanin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered (or recombinant) hosts can naturally express none or some(e.g., one or more, two or more, three or more, four or more, five ormore, or six or more) of the enzymes of the pathways described herein.Endogenous genes of the engineered hosts also can be disrupted toprevent the formation of undesirable metabolites or prevent the loss ofintermediates in the pathway through other enzymes acting on suchintermediates. Engineered hosts can be referred to as engineered hostcells, engineered cells, recombinant hosts recombinant host cells, orrecombinant cells. Thus, as described herein recombinant hosts caninclude nucleic acids encoding one or more of a dehydrogenase, adesaturase, a cytochrome P450, a decarboxylating thioesterase, asulphotransferase, a phosphotransferase, an acyl [acyl carrier protein(acp)] dehydrogenase, a dehydratase, or a hydratase as described in moredetail below.

In addition, the production of butadiene can be performed in vitro usingthe isolated enzymes described herein, using a lysate (e.g., a celllysate) from a host microorganism as a source of the enzymes, or using aplurality of lysates from different host microorganisms as the source ofthe enzymes.

4.1 Enzymes Generating the First Terminal Vinyl Group in theBiosynthesis of Butadiene

As depicted in FIGS. 2-6, the first vinyl group can be formed inpent-2-enoyl-[acp], 3-hydroxypropionyl-CoA, propanoyl-CoA, lactoyl-CoA,5-hydroxypentanoyl-CoA, butan-2-ol, 2-buten-1-ol, 2-buten-1-oldiphosphate, 4-hydroxy-pentanoyl-CoA, or 4-hydroxy pent-2-enoyl-CoA.

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in pent-2-enoyl-[acp] by anacyl-[acp]dehydrogenase such as the gene product of TcsD to produce 2,4-pentadienoyl-[acp]. (e.g., FIG. 2). The gene product of theacyl-[acp]dehydrogenase TcsD desaturates the terminal methylene ofpent-2-enoyl-[acp] to 2,4-pentadienoyl-[acp] (Mo et al., J Am. Chem.Soc., 2011, 133(4), 976-985).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in propanoyl-CoA, lactoyl-CoA, or3-hydroxypropionyl-CoA by butyryl-CoA dehydrogenase (EC 1.3.8.1),medium-chain acyl-CoA dehydrogenase (EC 1.3.8.7), 2-methylacyl-CoAdehydrogenase (EC 1.3.99.12), lactoyl-CoA dehydratase (EC 4.2.1.54) or3-hydroxypropionyl-CoA dehydratase (EC 4.2.1.116) to producepropenoyl-CoA. (e.g., FIG. 3).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is enzymatically formed in 5-hydroxypentanoyl-CoA (via5-hydroxy-pent-2-enoyl-CoA) by a 5-hydroxyvaleryl-CoA dehydratase (EC4.2.1.—). (e.g., FIG. 4). The dehydration of 5-hydroxyvalerate by5-hydroxyvaleryl-CoA dehydratase to 2,4-pentadienoyl-CoA has beencharacterized from Clostridium viride (Eikmanns and Buckel, Eur. J.Biochem., 1991, 197, 661-668).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is formed in 2-butanol (butan-2-ol) by a desaturase such asthe gene product of MdpJ to produce 3-buten-2-ol. (e.g., FIG. 5).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is formed in 2-buten-1-ol by an isomerase such as isolatedfrom Pseudomonas putida classified under EC 5.4.4.—to produce3-buten-2-ol. (e.g., FIG. 5) In some embodiments, the first vinyl groupleading to the synthesis of butadiene is formed in 2-buten-1-oldiphosphate by a 2-methyl-3-buten-2-ol synthase such as the gene productof Tps-MB01 to produce 3-buten-2-ol. (FIG. 5).

In some embodiments, the first vinyl leading to the synthesis ofbutadiene is formed in 4-hydroxy-pentanoyl-CoA by a dehydratase such aslinalool dehydratase classified in EC 4.2.1.127 or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1). (FIG. 6).

In some embodiments, the first vinyl group leading to the synthesis ofbutadiene is formed in 4-hydroxy-pent-2-enoyl-CoA by a dehydratase suchas linalool dehydratase classified in EC 4.2.1.127 or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1). (FIG. 6)

4.2 Enzymes Generating the Second Terminal Vinyl Group in theBiosynthesis of Butadiene

As depicted in FIG. 7, the second vinyl group can be enzymaticallyformed using a decarboxylating thioesterase, a decarboxylatingcytochrome P450 or a dehydratase.

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is formed by a decarboxylating thioesterase such as the geneproduct of CurM TE. (e.g., (i)-(iv) in FIG. 7).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is enzymatically formed by a decarboxylating cytochrome P450in the CYP152 family. (e.g., (v) in FIG. 7).

In some embodiments, the second vinyl group leading to the synthesis ofbutadiene is enzymatically formed by a dehydratase such as linalooldehydratase classified in EC 4.2.1.127 or a dehydratase classified underEC 4.2.1.—(such as one isolated from species such as Aquincolatertiaricarbonis or Methylibium petroleiphilum PM1). (e.g., (vi) in FIG.7).

Linalool may be regarded as 3-buten-2-ol substituted with an isohexenylR-group at the alpha position. The dehydration of linalool to myrcene isfavored thermodynamically and likely proceeds via deprotonation, wherethe R-group has no mechanistic role (Bordkorb et al., J. Biol. Chem.,2010, 285(40), 30436-30442).

4.3 Biochemical Pathways

4.3.1 Pathways Using 2,4-pentadienoyl-[acp] as Central Precursor toButadiene

In some embodiments, 2,4-pentadienoyl-[acp] is the central precursorleading to the synthesis of butadiene (FIG. 2).

In some embodiments, 2,4-pentadienoyl-[acp] can be produced by forming afirst vinyl group in pent-2-enoyl-[acp] using an acyl-[acp]dehydrogenasesuch as encoded by tcsD (FIG. 2) The pent-2-enoyl-[acp] can be producedby converting (R)-3-hydroxypentanoyl-[acp] using a3-hydroxyacyl-[acp]dehydratase classified under EC 4.2.1.59 such asencoded by fabZ (FIG. 2) The (R)-3-hydroxypentanoyl-[acp] can beproduced by converting (R)-3-oxopentanoyl-[acp] using a3-oxoacyl-[acp]reductase classified in EC 1.1.1.100 such as encoded byfabG or AnlG (FIG. 2) The (R)-3-oxopentanoyl-[acp] can be produced byconverting propanoyl-CoA using a β-ketoacyl-[acp]synthase such asencoded by tcsA and tcsB (FIG. 2). The propanoyl-CoA can be produced viaa number of pathways (FIG. 1).

In some embodiments, butadiene is synthesized from2,4-pentadienoyl-[acp] by conversion to (R)-3-hydroxypent-4-enoyl-[acp]by 3-hydroxyacyl-[acp]dehydratase classified under EC 4.2.1.59 such asencoded by fabZ (FIG. 2); followed by conversion to3-sulphorylpent-4-enoyl-[acp] by sulphotransferase classified under EC2.8.2.—such as encoded by CurM ST or OLS ST (FIG. 7); followed byconversion to butadiene by decarboxylating thioesterase such as encodedby CurM TE. (FIG. 7).

In some embodiments, butadiene is synthesized from2,4-pentadienoyl-[acp] by conversion to (R)-3-hydroxypent-4-enoyl-[acp]by 3-hydroxyacyl-[acp]dehydratase classified under EC 4.2.1.59 such asencoded by fabZ (FIG. 2); followed by conversion to3-phosphopent-4-enoyl-[acp] by phosphotransferase classified under EC2.7.1.—(FIG. 7); followed by conversion to butadiene by decarboxylatingthioesterase such as encoded by CurM TE. (FIG. 7).

4.3.2 Pathways Using Propenoyl-CoA as Central Precursor to Butadiene

In some embodiments, propenoyl-CoA is the central precursor leading tothe synthesis of butadiene (FIG. 3).

In some embodiments, propenoyl-CoA is synthesized from propanoyl-CoA bybutyryl-CoA dehydrogenase (classified under EC 1.3.8.1), medium-chainacyl-CoA dehydrogenase (classified under EC 1.3.8.7) or 2-methylacyl-CoAdehydrogenase (classified under EC 1.3.99.12). (e.g., FIG. 3).

In some embodiments, propenoyl-CoA is synthesized from the centralmetabolite, pyruvate, by conversion of pyruvate to L-lactate byL-lactate dehydrogenase (classified under EC 1.1.1.27); followed byconversion to lactoyl-CoA by proprionate CoA-transferase (classifiedunder EC 2.8.3.1); followed by conversion to propenoyl-CoA bylactoyl-CoA dehydratase (classified under EC 4.2.1.54). (e.g., FIG. 3).

In some embodiments, propenoyl-CoA is synthesized from the centralmetabolite, malonyl-CoA, by conversion to conversion to3-hydroxypropionate by 3-hydroxypropionate dehydrogenase (classifiedunder EC 1.1.1.59); followed by conversion to 3-hydroxypropionyl-CoA by3-hydroxyisobutyryl-CoA hydrolase (classified under EC 6.2.1); followedby conversion to propenoyl-CoA by 3-hydroxypropionyl-CoA dehydratase(classified under EC 4.2.1.116). (e.g., FIG. 3).

In some embodiments, butadiene is synthesized from propenoyl-CoA byconversion to 3-oxopent-4-enoyl-CoA by β-ketothiolase such as thatclassified under EC 2.3.1.16; followed by conversion to(R)-3-hydroxypent-4-enoyl-CoA by acetoacetyl-CoA reductase (classifiedunder EC 1.1.1.36) such as the gene product of phaB (FIG. 3); followedby conversion to 3-sulphorylpent-4-enoyl-CoA by sulphotransferaseclassified under EC 2.8.2.—such as encoded by CurM ST or OLS ST;followed by conversion to butadiene by decarboxylating thioesterase suchas encoded by CurM TE. (FIG. 7).

In some embodiments, butadiene is synthesized from propenoyl-CoA byconversion to 3-oxopent-4-enoyl-CoA by β-ketothiolase such as thatclassified under EC 2.3.1.16 (FIG. 3); followed by conversion to(R)-3-hydroxypent-4-enoyl-CoA by acetoacetyl-CoA reductase (classifiedunder EC 1.1.1.36) such as the gene product of phaB (FIG. 3); followedby conversion to 3-phosphopent-4-enoyl-CoA by phosphotransferaseclassified under EC 2.7.1.—(FIG. 7); followed by conversion to butadieneby decarboxylating thioesterase such as encoded by CurM TE. (FIG. 7).

4.3.2 Pathway Using 2,4-pentadienoyl-CoA as Central Precursor toButadiene

In some embodiments, 2,4-pentadienoyl-CoA is the central precursorleading to the synthesis of butadiene (FIG. 4).

In some embodiments, 2,4-pentadienoyl-CoA is synthesized from5-aminovalerate (5-aminovaleric acid) by conversion to 5-oxopentanoate(5-oxopentanoic acid) by a 5-aminovalerate transaminase (classifiedunder EC 2.6.1.48); followed by conversion to 5-hydroxypentanoate by a5-hydroxyvalerate dehydrogenase such as the gene product of cpnD or adehydrogenase from Clostridium viride; followed by conversion to5-hydroxypentanoyl-CoA by a 5-hydroxypentanoate CoA-transferase(classified under EC 2.8.3.14); followed by conversion to2,4-pentadienoyl-CoA by a 5-hydroxyvaleryl-CoA dehydratase (classifiedunder EC 4.2.1.—) (e.g., that from Clostridium viride). (e.g., FIG. 4).

In some embodiments, 2,4-pentadienoyl-CoA is synthesized from levulinicacid by conversion to levulinyl-CoA by CoA-ligase (classified under EC6.2.1.—); followed by conversion to 4-hydroxypentanoyl-CoA by secondaryalcohol dehydrogenase (classified under EC 1.1.1.B4) or3-hydroxybutanoate oxidoreductase (classified under EC 1.1.1.30);followed by conversion to 4-hydroxypent-2-enoyl-CoA by reversibletrans-2-enoyl-CoA reductase (classified under EC 1.3.1.-(8,38,44);followed by conversion to 2,4-pentadienoyl-CoA by a dehydratase such aslinalool dehydratase (classified in EC 4.2.1.127) or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1). (FIG. 6).

In some embodiments, butadiene is synthesized from 2,4-pentadienoyl-CoAby conversion to 4-pentenoyl-CoA(pent-4-enoyl-CoA) by5-hydroxyvaleryl-CoA dehydratase (classified under EC 1.3.1.44) (e.g.,that from Clostridium viride) (FIG. 4); followed by conversion tobutadiene by decarboxylating cytrochrome P450 in the CYP152 family.(FIG. 7).

4.3.4 Pathway Using Pent-4-enoyl-CoA as Central Precursor to ButadieneWithout First Forming 2,4-pentadienoyl-CoA

In some embodiments, pent-4-enoyl is the central precursor leading tothe synthesis of butadiene without first forming 2,4-pentadienoyl-CoA.

In some embodiments, pent-4-enoyl-CoA is synthesized from levulinic acidby conversion to levulinyl-CoA by a CoA ligase (classified under EC6.2.1-); followed by conversion to 4-hydroxy-pentanoyl-CoA by asecondary alcohol dehydrogenase classified under EC 1.1.1.B4 such or a3-hydroxy butanoate oxidoreductase classified under EC 1.1.1.30;followed by conversion to pentanoyl-4-enoyl-CoA by a dehydratase such aslinalool dehydratase (classified in EC 4.2.1.127) or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1). (FIG. 6).

In some embodiments, butadiene is synthesized from 4-pentenoyl-CoA(pent-4-enoyl-CoA) by decarboxylating cytrochrome P450 in the CYP152family. (FIG. 7).

4.3.5 Pathway Using 3-Buten-2-Ol as Central Precursor to Butadiene

In some embodiments, 3-buten-2-ol is the central precursor leading tothe synthesis of butadiene. (FIG. 5).

In some embodiments, 2-buten-1-ol is synthesized from crotonyl-CoA byconversion to crotonic acid by a succinate-CoA ligase (classified underEC 6.2.1.5); followed by conversion to 2-buten1-al by along-chain-aldehyde dehydrogenase (classified under EC 1.2.1.48);followed by conversion to 2-buten-1-ol by an allyl-alcohol dehydrogenase(classified under EC 1.1.1.54). See FIG. 5.

In some embodiments, 3-buten-2-ol is synthesized from 2-buten-1-ol byconversion to 2-buten-1-ol phosphate by a mevalonate kinase (classifiedunder EC 2.7.1.36); followed by conversion to 2-buten-1-ol diphosphateby a phosphomevalonate kinase (EC 2.7.4.2); followed by conversion to3-buten-2-ol by a 2-methyl-3-buten-2-ol synthase such as that encoded byTps-MBO1. (e.g., FIG. 5).

In some embodiments, 3-buten-2-ol is synthesized from 2-buten-1-ol byconversion to 2-buten-1-ol diphosphate by a diphosphotransferases suchas a thiamine diphosphokinase (classified under EC 2.7.6.2); followed byconversion to 3-buten-2-ol by a 2-methyl-3-buten-2-ol synthase such asencoded by Tps-MBO1. (e.g., FIG. 5).

In some embodiments, 3-buten-2-ol is synthesized from the centralmetabolite, pyruvate, by conversion of pyruvate to 2-acetolactate by anacetolactate synthase (classified under EC 2.2.1.6); followed byconversion to (R)-acetoin by an acetolactate decarboxylase (classifiedunder EC 4.1.1.5); followed by conversion to 2,3 butanediol by a(R,S)-butanediol dehydrogenase such as encoded by budC; followed byconversion to butanone (butan-2-one) by a propanediol dehydratase(classified under EC 4.2.1.28); followed by conversion to 2-butanol(butan-2-ol) by a (R)-specific secondary alcohol dehydrogenase(classified under EC 1.1.1.B4); followed by conversion to 3-buten-2-olby a desaturase or a monooxygenase such as the gene product of MdpJ orcytochrome P450 in, for example, the CYP4 family. (e.g., FIG. 5).

In some embodiments, 3-buten-2-ol is synthesized from the centralprecursor, propanoyl-CoA, by conversion of propanoyl-CoA to3-oxopentanoyl-CoA using a β-ketothiolase classified under EC 2.3.1.16;followed by conversion to 3-oxopentanoate by thioesterase classifiedunder EC 3.1.2.—such as the gene product of YciA, tesB, tesA or fadM;followed by conversion to 2-butanone (butan-2-one) by acetoacetatedecarboxylase classified under EC 4.1.1.4; followed by conversion to2-butanol (butan-2-ol) by a (R)-specific secondary alcohol dehydrogenase(EC 1.1.1.B4); followed by conversion to 3-buten-2-ol by a desaturase ora monooxygenase such as the gene product of MdpJ or cytochrome P450 in,for example, the CYP4 family. (e.g., FIG. 5).

In some embodiments, 3-buten-2-ol is synthesized from the centralprecursor, propenoyl-CoA, by conversion of propenoyl-CoA to3-oxopent-4-enoyl-CoA using a β-ketothiolase classified under EC2.3.1.—(such as that classified under EC2.3.1.16); followed byconversion to 3-oxopent-4-enoate by thioesterase classified under EC3.1.2.—such as the gene product of YciA, tesB, tesA or fadM; followed byconversion to 3-buten-2-one by acetoacetate decarboxylase classifiedunder EC 4.1.1.4; followed by conversion to 3-buten-2-ol by a(R)-specific secondary alcohol dehydrogenase (EC 1.1.1.B4). (e.g., FIG.5).

In some embodiments, 3-buten-2-ol is synthesized from 2-buten-1-ol by anisomerase classified under EC 5.4.4.—such as that isolated fromPseudomonas putida. (e.g., FIG. 5).

In some embodiments, butadiene is synthesized from 3-buten-2-ol bylinalool dehydratase in enzyme class EC 4.2.1.127 or a dehydrataseclassified under EC 4.2.1.—(such as one isolated from species such asAquincola tertiaricarbonis or Methylibium petroleiphilum PM1). (e.g.,FIG. 7).

4.4 Cultivation Strategy

In some embodiments, butadiene is biosynthesized in a recombinant hostusing a fermentation strategy that can include anaerobic, micro-aerobicor aerobic cultivation of the recombinant host.

Pathways in the synthesis of butadiene that incorporate enzymesrequiring molecular oxygen and enzymes characterized in vitro as beingoxygen sensitive require a micro-aerobic cultivation strategymaintaining a low dissolved oxygen concentration, whilst maintainingsufficient oxygen transfer to prevent substrate oxidation controlledconditions (Chayabatra & Lu-Kwang, Appl. Environ. Microbiol., 2000,66(2), 493 0 498).

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes is employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation inthe synthesis of butadiene.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of butadiene derives from biological or non-biologicalfeedstocks.

In some embodiments, the biological feedstock is, includes, or derivesfrom, monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin such as levulinic acid and furfural, lignin,triglycerides such as glycerol and fatty acids, agricultural waste ormunicipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90, 885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011,155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009,139, 61-67).

The efficient catabolism of lignin-derived aromatic compounds suchbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794). The efficient utilization ofagricultural waste, such as olive mill waste water has been demonstratedin several microorganisms, including Yarrowia lipolytica (Papanikolaouet al., Bioresour. Technol., 2008, 99(7), 2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104,155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172;Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5),647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).

In some embodiments, the non-biological feedstock is, or derives from,natural gas, syngas, CO₂/H₂, methanol, ethanol, non-volatile residue(NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses.

The efficient catabolism of methanol has been demonstrated for themethylotropic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and 11₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15), 5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1), 152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be from the genus Escherichia such asEscherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be sources of genes toconstruct recombinant host cells described herein that are capable ofproducing butadiene.

In some embodiments, the host microorganism is a eukaryote. Eukaryotescan be, for example, fungi (e.g., filamentous fungi or yeasts). Forexample, the eukaryote can be from the genus Aspergillus such asAspergillus niger; from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingbutadiene.

4.5 Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten, ormore of such steps. Where less than all the steps are included in such amethod, the first step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined in section 4.3are the result of enzyme engineering via non-direct or rational enzymedesign approaches with to aims of improving activity, improvingspecificity, reducing feedback inhibition, reducing repression,improving enzyme solubility, changing stereo-specificity, or changingco-factor specificity.

In some embodiments, the enzymes in the pathways outlined in section 4.3are gene dosed, i.e., overexpressed, into the resulting geneticallymodified organism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis are utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to butadiene.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data areutilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to butadiene.

In some embodiments requiring intracellular availability ofpropanoyl-CoA or propenoyl-CoA for butadiene synthesis, genes (e.g.,endogenous genes) encoding enzymes catalyzing the hydrolysis ofpropionyl-CoA and acetyl-CoA can be attenuated in the host organism.

In some embodiments requiring the intracellular availability ofpropanoyl-CoA or propenoyl-CoA for butadiene synthesis, genes (e.g.,endogenous genes) encoding enzymes consuming propanoyl-CoA via themethyl-citrate cycle can be attenuated in the host organism (Upton andMckinney, Microbiology, 2007, 153, 3973-3982).

In some embodiments requiring the intracellular availability ofpropanoyl-CoA or propenoyl-CoA for butadiene synthesis, genes (e.g.,endogenous genes) encoding enzymes consuming propanoyl-CoA to pyruvatecan be attenuated in the host organism.

In some embodiments requiring the intracellular availability ofpropanoyl-CoA or propenoyl-CoA for butadiene synthesis, genes (e.g.,endogenous genes) encoding enzymes consuming propanoyl-CoA tomalonyl-CoA can be attenuated in the host organism.

In some embodiments requiring the intracellular availability ofpropanoyl-CoA or propenoyl-CoA via L-threonine as central metabolite forbutadiene synthesis, a feedback-resistant threonine deaminase isgenetically engineered into the host organism (Tseng et al., MicrobialCell Factories, 2010, 9:96).

In some embodiments requiring condensation of acetyl-CoA andpropanoyl-CoA/propenoyl-CoA for butadiene synthesis, the genes (e.g.,endogenous genes) encoding β-ketothiolases catalyzing the condensationof acetyl-CoA to acetoacetyl-CoA (such as the AtoB or phaA genes) can beattenuated.

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, the genes (e.g., endogenous genes) encodingpolymer synthase enzymes can be attenuated in the host strain.

In some embodiments requiring the intracellular availability ofacetyl-CoA for butadiene synthesis, a host that is deficient (e.g.,attenuated level of activity) in one or more enzymes in the acetatesynthesis pathway can be used. For example, a host that is deficient ina phosphotransacetylase (encoded by the pta gene) can be used (Shen etal., Appl. Environ. Microbio., 2011, 77(9), 2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for butadiene synthesis, a gene (e.g., an endogenous gene) inan acetate synthesis pathway encoding an acetate kinase, such as ack,can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for butadiene synthesis, a gene (e.g., an endogenous gene)encoding an enzyme catalyzing the degradation of pyruvate to lactate,such as ldhA, can be attenuated (Shen et al., Appl. Environ. Microbio.,2011, 77(9), 2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for butadiene synthesis, a gene (an endogenous gene) encodingan enzyme catalyzing the degradation of phophoenolpyruvate to succinate,such as frdBC, can be attenuated (see, e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA for butadiene synthesis, a gene (e.g., an endogenous gene)encoding an enzyme catalyzing the degradation of acetyl-CoA to ethanol,such as adhE, can be attenuated (Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofL-glutamate for butadiene synthesis, the genes (e.g., endogenous genes)encoding enzymes catalyzing anaplerotic reactions supplementing thecitric acid cycle intermediates can be amplified.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of butadiene, a puridine nucleotide transhydrogenase gene,such as UdhA, can be overexpressed in the host organisms (Brigham etal., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of butadiene, a glyceraldehyde-3P-dehydrogenase gene suchas GapN can be overexpressed in the host organisms (Brigham et al.,2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of butadiene, a malic enzyme gene, such as maeA or maeBcan be overexpressed in the host organisms (Brigham et al., 2012,supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of butadiene, a glucose-6-phosphate dehydrogenase genesuch as zwf can be overexpressed in the host organisms (Lim et al.,Journal of Bioscience and Bioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of butadiene, a fructose 1,6 diphosphatase gene such asfbp can be overexpressed in the host organisms (Becker et al., Journalof Biotechnology, 2007, 132, 99-109).

In some embodiments, the efflux of butadiene across the cell membrane tothe extracellular media can be enhanced or amplified by geneticallyengineering structural modifications to the cell membrane or increasingany associated transporter activity for butadiene.

In some embodiments, genes (e.g., endogenous genes) encoding oxygenasesdegrading butadiene to toxic intermediates such as 1,2-epoxy-3-buteneand 1,2:3,4-diepoxybutane can be attenuated in the host organism (see,e.g., Sweeney et al., Carcinogenesis, 1997, 18(4), 611-625).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Enzyme Activity of Linalool Dehydratase Using3-Buten-2-Ol as Substrate

The his-tagged linalool dehydratase (EC 4.2.1.127) from Castellanielladefragrans was cloned into a pARZ4 vector and transformed into E. coliBL21. The resulting strain was cultivated and induced using 1M IPTG(isopropylthio-β-galactoside) in a shake flask culture containing LuriaBroth media and kanamycin selection pressure.

The cells from each of the induced shake flask cultures were harvestedand pelleted by centrifugation. The cell pellet was resuspended and thecells were lysed. The cell debris was separated from the supernatant viacentrifugation and filtered using a 0.2 μm filter. The enzyme waspurified from the filtered supernatant using Ni-affinity chromatographyand concentrated and buffer exchanged using a Vivaspin 15R CentrifugalConcentrator and Hi-trap Desalting column into 80 mM Tris buffer (pH=9).

Non-native enzyme activity assays were undertaken in a buffer containing11 mM of 3-buten-2-ol at 25° C. The activity assays were undertaken in 2mL septum-sealed vials, thereby allowing butadiene accumulation in theheadspace. The reaction was initiated by adding 1 mL of purified enzymeto the assay buffer containing the substrate.

The headspace was sampled for butadiene analysis by GC-MS (gaschromatography-mass spectrometry). The retention time for the butadienestandard and the assay samples were within 2%. The ratio of the MS ionpeak areas from the butadiene standard and the MS ion peak areas of thesamples agree to within 20%. Also, the ion peak areas were above thelimit of quantitation for the GC-MS.

These findings show that linalool dehydratase (EC 4.2.1.127) accepts3-buten-2-ol as a substrate, thereby synthesizing butadiene.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of butadiene synthesis, the method comprising introducing afirst vinyl group into a first vinyl group acceptor compound using adehydratase, a dehydrogenase, an isomerase, a synthase or a desaturase.2. A method of butadiene synthesis, the method comprising introducing asecond vinyl group into a second vinyl group acceptor compound using adecarboxylating thioesterase, a decarboxylating cytochrome P450 or adehydratase.
 3. The method of claim 2, further comprising, prior to theintroduction of the second vinyl group, introducing a first vinyl groupinto a first vinyl group acceptor compound to produce the second vinylgroup acceptor compound, using a dehydratase, a dehydrogenase, anisomerase, a synthase or a desaturase.
 4. The method according to claim2, wherein the decarboxylating thioesterase introducing the second vinylgroup is an engineered enzyme having greater than 70% homology to thedecarboxylating thioesterase from Lyngbya majuscula (CurM TE),Pseudomonas entomophila, H. ochraceum, Synechococcus PCC 7002,Cyanothece PCC 7424 or Cyanothece PCC 7822; the decarboxylatingcytochrome P450 introducing the second vinyl group is an engineeredenzyme having greater than 70% homology to the decarboxylatingcytochrome P450 from Jeotgalicoccus sp. ATCC 8456; or the dehydrataseintroducing the second vinyl group is an engineered enzyme havinggreater than 70% homology to linalool dehydratase (EC 4.2.1.127) fromCastellaniella defragrans.
 5. The method according to claim 2, where thedecarboxylating thioesterase introducing the second vinyl groupcatalyses the hydrolysis of either 3-sulphorylpent-4-enoyl-[acp],3-phosphopent-4-enoyl-[acp], 3-sulphorylpent-4-enoyl-CoA or3-phosphopent-4-enoyl-CoA.
 6. The method of claim 5, wherein, followingthe introduction of the second vinyl group, the resulting compoundundergoes spontaneous decarboxylation to butadiene. 7-8. (canceled) 9.The method according to claim 3, wherein the dehydratase (EC 4.2.1.—)enzyme introducing the first vinyl group is an engineered enzyme havinggreater than 70% homology to the 5-aminovaleryl-CoA dehydratase from C.viride, linalool dehydratase (EC 4.2.1.127) or a dehydratase (EC4.2.1.—) from species such as Aquincola tertiaricarbonis or Methylibiumpetroleiphilum PM1.
 10. The method according to claim 1, wherein theacyl-ACP dehydrogenase introducing the first vinyl group is anengineered enzyme having greater than 70% homology to the gene productof tcsD; the desaturase/monooxygenase introducing the first vinyl groupis an engineered enzyme have greater than 70% homology to the geneproduct of MdpJ or cytochrome P450 CYT4 family; the synthase introducingthe first vinyl group is an engineered enzyme have greater than 70%homology to 2-methyl-3-buten-2-ol synthase encoded by Tps-MBO1; or theisomerase introducing the first vinyl group is an engineered enzymehaving greater than 70% homology to the isomerase from Pseudomonasputida catalyzing the conversion of 2-methyl-3-buten-2-ol to2-methyl-3-buten-1-ol. 11-13. (canceled)
 14. The method according toclaim 2, wherein the decarboxylating thioesterase converts3-sulphorylpent-4-enoyl-[acp] or 3-phosphopent-4-enoyl-[acp] assubstrate to butadiene.
 15. The method according to claim 2, wherein thedecarboxylating cytochrome P450 converts pent-4-enoic acid to butadiene.16. The method according to claim 15, wherein the hydrogen peroxideco-substrate required for the conversion of pent-4-enoic acid tobutadiene is provided by the activity of a primary amine oxidase. 17.The method according to claim 2, wherein the dehydratase converts3-buten-2-ol to butadiene.
 18. The method according to claim 1, whereinthe isomerase converts 2-buten-1-ol to 3-buten-2-ol.
 19. The method ofclaim 1, wherein the method comprises a fermentation process using ahost cell expressing an enzyme that catalyzes the introduction of afirst vinyl group, an enzyme that catalyzes the introduction of a secondvinyl group, or one or two enzymes that catalyze the introduction of afirst and a second vinyl group.
 20. (canceled)
 21. The method accordingto claim 19, wherein the host cell is a prokaryote is of the genusEscherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas,Bacillus or Rhodococcus or a eukaryote of the genus Aspergillus,Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula orKluyveromyces.
 22. (canceled)
 23. The method according to claim 19,wherein the fermentation process comprises anaerobic, micro-aerobic oraerobic cell cultivation.
 24. The method according to claim 19, whereincell retention strategies using, for example, ceramic hollow fibremembranes are employed to achieve and maintain a high cell densityduring fermentation.
 25. The method according to claim 19, wherein theprincipal carbon source fed to the fermentation derives from biologicalor non-biological feedstocks.
 26. The method according to claim 25,where the biological feedstock is, or derives from, monosaccharides,disaccharides, hemicellulose such as levulinic acid and furfural,cellulose, lignocellulose, lignin, triglycerides such as glycerol andfatty acids, agricultural waste or municipal waste.
 27. The methodaccording to claim 25, where the non-biological feedstock is, or derivesfrom, natural gas, syngas, CO₂/H₂, methanol, ethanol, non-volatileresidue (NVR), caustic wash from a cyclohexane oxidation processes, orother waste stream from the chemical or petrochemical industries.